The central hypothesis of this application is that macromolecular diffusion in brain extracellular space (ECS) is controlled by molecular configuration, local architecture, the extracellular matrix, and bulk flow. We will use the Integrative Optical Imaging (IOI) technique support b y the TMA+ method. In the IOI method, a diffusing cloud of fluorescent macromolecules is imaged with a compound microscope and quantified. In the TMA+ method, the concentration of tetramethylammonium ions is measured using ion-selective microelectrodes. Hardware and software developments during the last funding period have greatly improved both techniques. Rat brain slices w3ill be the main preparation but a consultant will provide in vivo data. There are four Specific Aims: Aim 1. How does ECS volume interact with the shape and size of a macromolecule to affect diffusion? We have shown that some large globular molecules diffuse very differently from linear polymers of similar molecular weight. We will analyze the diffusion behavior of several new macromolecules and then alter the ECS by osmotic manipulation and by using a slice preparation that mimics ischemia to determine the relation between ECS size and molecular size. Aim 2. How much does brain architecture channel substances? Does brain structure channel substances preferentially in one direction rather than another (anisotropy) or pose local barriers to the movement of substances (inhomogeneity)? Aim 3. How much does the extracellular matrix affect diffusion? The actual amount and distribution of the extracellular matrix are far from clear. Having established how various other factors affect diffusion in Aims 1-2 we will be in a position to tackle this important but difficult problem. Aim 4. How important is bulk flow in the transport of material in the ECS? Bulk flow could move substances in a specific direction over long distances but conclusive evidence is lacking. Dr Abbott (consultant, London) has new data and we have sophisticated image analysis software, so we will try to settle this long-standing issue. The origin of bulk flow is addressed in a model developed by Dr Patlak (consultant, Stony Brook). This application is focused on basic research with especial importance for volume transmission but many of the results will be relevant to clinical issues, especially drug delivery.